1. Field of the Invention
The present invention relates to a method which uses a microorganism for producing a substance. In the present invention, the microorganism is typically a bacteria belonging to the genus Escherichia, or coryneform bacteria, which are conventionally used for production of substances. The substance to be produced may be selected from those conventionally produced by using microorganisms, for example, L-amino acids, nucleic acids, antibiotics, vitamins, growth factors, physiologically active substances, and so forth. The present invention discloses means for improving production of final target substances in methods using microorganisms for producing substances.
2. Description of the Related Art
Many of organisms acquire energy required for survival and function by respiration. In the respiration of microorganisms, the function of various enzyme complexes is generally dependent on the species or growth environment, and energy acquisition efficiency also can vary significantly. Carbohydrates, proteins and aliphatic acids are converted into acetyl-CoA by the glycolysis, β-oxidation, and so forth, and decomposed in the citric acid cycle. Then, the energy preserved in the form of NADH is used for proton excretion from microbial cells with the aid of NADH dehydrogenase (NDH), and an electron transfer system consisting of oxidoreductases, and thereby a proton concentration gradient, is formed between the inside and outside of the cytoplasmic membrane. This proton concentration gradient is the driving force of adenosine triphosphate (ATP) synthesis. At this time, pathways of electron transfer include pathways showing high and low proton excretion ability, depending on the combination of NDH and oxidoreductases. It is thought that a pathway of high proton excretion ability shows high energy efficiency and a pathway of low proton excretion ability shows low energy efficiency. Thus, one kind of microorganism simultaneously contains a plurality of respiratory chain electron transfer pathways in parallel, and those pathways include those of high energy efficiency and low energy efficiency.
Two kinds each of NDHs and terminal oxidases exist in the respiratory chain of Escherichia coli under aerobic conditions. That is, NDH-1, encoded by the nuo operon, is known to have high energy efficiency, and NDH-II, encoded by ndh, is known to have low energy efficiency. Furthermore, cytochrome bo-type oxidase, encoded by the cyoABCD operon, and classified as a SoxM type (Castresana, J. and Saraste, M., Trends in Biochem. Sci, 20, 443-448 (1995)) is known to show high energy efficiency, and cytochrome bd-type oxidase, encoded by cydAB, is known to show low energy efficiency. Although it is known that the levels of expression of these respiratory chain enzymes vary in response to their growth environment (Minagawa et al., The Journal of Biological Chemistry, 265:11198-11203 (1990); Tseng et al., Journal of Bacteriology, 178:1094-1098 (1996); Green et al., Molecular Microbiology, 12:433-444 (1994); Bongaerts et al., Molecular Microbiology, 16:521-534 (1995)), much is unknown about the physiological meaning of their expression patterns.
Furthermore, Corynebacterium glutamicum contains a cytochrome bc1 complex and at least two kinds of terminal oxidases, SoxM type oxidase and cytochrome bd type oxidase (The Second Symposium Concerning Metabolic Engineering, Lecture Abstracts, 1999). This shows that there are two kinds of electron transfer pathways from a quinone pool to an oxygen molecule and include a pathway utilizing cytochrome bc1 complex and SoxM type oxidase, and a pathway utilizing only the cytochrome bd type oxidase. It is thought that the former is an electron transfer pathway of high energy efficiency in which the proton transfer value for transfer of one electron is high, and the latter is an electron transfer pathway of low energy efficiency in which proton transfer value for transfer of one electron is low.
As for the terminal oxidase of E. coli, in a comparison of growth yields in aerobic cultures of a mutant strain having only the cytochrome bo-type oxidase, a mutant strain having only the cytochrome bd-type oxidase, and a wild-type strain having both, the growth yield will be the lowest in the mutant strain having only the cytochrome bd-type oxidase, and it depends on the kind and energy acquisition efficiency of terminal oxidase (Annual Meeting of the Society for fermentation and Bioengineering Japan, 1995, Lecture Abstracts, No. 357).
Furthermore, the energy efficiency of deficient mutants of some respiratory chain enzymes has been reported (Calhoun et al., Journal of Bacteriology, 17.5:3020-3925 (1993)).
However, there have been no reports concerning a change in energy efficiency by amplification of a respiratory chain gene providing high efficiency such as those for NDH-I and SoxM type oxidase, and an attempt to utilize such for production of substances has also not been reported. Furthermore, no attempts have been made to delete a respiratory chain enzyme of low efficiency such as NDH-II and cytochrome bd-type oxidase for production of substances.